Date of Award

1-1-2011

Degree Name

Doctor of Philosophy

Department

Biological Sciences

First Advisor

Dr. Silvia Rossbach

Second Advisor

Dr. Maria Scott

Third Advisor

Dr. Todd Barkman

Fourth Advisor

Dr. Elke Schoffers

Abstract

"The rhizopine concept suggests that rhizopines, which are inositol derivatives, function as nutritional mediators and play an important role in host-bacteria interactions. This thesis analyzes the hypothesis that not only rhizopines but also naturally occurring inositol isomers, such as myo-, D-chiro-, and scyllo -inositol, function as nutritional mediators in the Sinorhizobium meliloti - alfalfa nitrogenfixing symbiosis. The idhA-encoded myoinositol dehydrogenase is responsible for the first step in the degradation of myo- and D-c/z/ra-inositol. A different dehydrogenase, IolY, was found to be essential for growth with scyllo-'mositol. The iolA and iolCDEB genes were required for the further degradation of myo-, D-chiroand scyllo-'mositol. The idhA and iolA genes are monocistronically transcribed, while the iolYRCDEB genes comprise an operon. The gene product of iolR, an RpiR-like repressor, negatively regulates iol gene expression in the absence of inositol. IolR was shown to recognize a conserved binding motif (5'-GGAA5-nTTCC-3') in the promoter regions of the idhA and iolY genes. Two additional IolR-binding motifs were confirmed within the iolYRCDEB operon, upstream of iolR and iolC, indicating that the operon contains two internal operators and that IolR has autoregulatory function. IolR-mediated repression was antagonized by a pathway intermediate, probably 2-keto-5-deoxy-gluconic acid 6-phosphate. The iolA gene was not regulated by IolR, but constitutively expressed. The iolA gene product does not seem to be part of the central inositol catabolic pathway but rather a point of intersection of different metabolic pathways, including valine catabolism. The inositol derivative scylloinosamine (SIA) has been proposed to be a rhizopine. We used chemically synthesized SIA to confirm its proposed structure. The same genes essential for the degradation of crude rhizopine isolated from root nodules, namely the idhA and mocABCR genes were also found to be required for the utilization of the chemically synthesized SIA. In plant-bacteria interactions, mutants with insertions in the idhA, iol A, the individual iolYRCDE and mocACR genes could not compete with the wild type in a nodule occupancy assay on alfalfa plants. In conclusion, this work strongly supports the hypothesis that rhizopine and inositol metabolism are important nutritional and signaling factors in the S. meliloti - alfalfa symbiosis."

Comments

5th Advisor: Dr. David Huffman

Access Setting

Dissertation-Open Access

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